Peptide amidating activity in human bronchoalveolar lavage fluid

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.     

Abortion in foxhounds and a ewe flock associated with Salmonella montevideo infection

Salmonella montevideo is a recognised cause of ovine abortion and can cause disease in other domestic animals and humans. The organism was isolated from the aborted fetuses of a bitch from a pack of foxhounds. The subsequent collection of rectal swabs from the foxhounds at approximately two week intervals over a 48-day period resulted in the isolation of S montevideo from 50 of the 61 hounds in the pack on one or more occasions. Some of the hounds had gained access to an open pit containing dead ewes and aborted fetuses on a farm where the housed ewe flock was experiencing S montevideo infection. The S montevideo isolates from both the ovine and canine samples had a plasmid of 120 kilobases with an identical restriction endonuclease fragmentation pattern. The only other isolate from a contemporary outbreak lacked this plasmid. It was concluded that this case offered further evidence of the potential for salmonella infection to be spread by the scavenging of carcasses.     

A Golgi localization signal identified in the Menkes recombinant protein

Menkes disease arises from a genetic impairment in copper transport. The gene responsible for the phenotype has been identified as a copper transporting ATPase ( ATP7A ). Recently, the protein encoded by the ATP7A gene has been localized to the Golgi complex. In order to investigate the role of the Menkes disease protein in copper transport, recombinant constructs containing both the full-length open reading frame and an alternatively spliced form have been successfully expressed and localized in mammalian cells. Other studies of a patient with occipital horn syndrome, an allelic variant of Menkes disease, have demonstrated that only this alternatively spliced isoform and not the full-length form is expressed in this patient. The milder form of this patient's phenotype suggests that the alternatively spliced isoform has some functional role in copper transport. In the present study the full-length recombinant Menkes protein was shown by immunofluorescence to localize to the Golgi apparatus and the alternatively spliced form, lacking sequences for transmembrane domains 3 and 4 encoded by exon 10, was shown to localize to the endoplasmic reticulum. Using sequences from exon 10 fused to a non-Golgi reporter molecule, a 38 amino acid sequence containing transmembrane domain 3 of the Menkes protein was found to be sufficient for localization to the Golgi complex. Therefore, the protein sequence encoded by exon 10 may be responsible for this differential localization and both isoforms may be required for comprehensive transport of copper within the cell.     

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.     

Inhibition of ingestive behavior following fourth ventricle bombesin injection in chronic decerebrate rats.

Tested the hypothesis that the effects of 4th ventricle bombesin (BN) injection on feeding require interaction with forebrain neural systems by measuring intraoral sucrose (0.1 M) in tube-fed control and tube-fed supracollicular decerebrate rats after 4th ventricle injections of 1, 5, 10, 20, and 50 ng BN. Fourth ventricle injections of all doses of BN reliably suppressed sucrose intake in both control and chronic decerebrate rats. These results indicate that caudal brain-stem afferent signals produced by 4th ventricle BN injections are integrated by the local neural circuitry of the caudal brain stem, independent of the forebrain systems, to modulate ingestive behavior. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Nucleic acid delivery: the missing pieces of the puzzle?

The delivery of genes or RNA interference (RNAi) agents can increase or decrease the expression of virtually any protein in a cell, and this process opens the path for cures to most diseases that afflict humans. However, the high molecular weight, anionic nature, and instability of nucleic acids in the presence of enzymes pose major obstacles to their delivery and frustrates their use as human therapies. This Account describes current ideas about the mechanisms in nonviral nucleic acid delivery and how lipidic and polymeric carriers can overcome some of the critical barriers to delivery. Over the last 20 years, researchers have developed a multitude of polymeric and lipidic vectors, but only a small fraction of these have progressed into clinical trials. None of these vectors has received FDA approval, which indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. However, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery, and an even smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies are needed to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane. siRNA delivery involves fewer barriers. siRNA is more readily released from the carrier and more resistant to enzymatic degradation, and its target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RNA-induced silencing complex (RISC) formation and activity is unknown, which makes specific targeting of the RISC for more efficient delivery difficult. Furthermore, we would like to identify the factors that favor the binding of siRNA to Ago-2. If we could understand how the half-life of siRNA and Ago-2/siRNA complex in the cytoplasm can be modulated without interfering with RISC functions that are essential for normal cell activity, we could increase siRNA delivery efficiency. In this Account, we review the current synthetic vectors and propose alternative strategies in a few cases. We also suggest how certain cellular mechanisms might be exploited to improve gene transfection and silencing. Finally, we discuss whether some carriers that deliver the siRNA to cells could also repackage the siRNA into exosomes. The exosomes would then transport the siRNA into a subsequent population of cells that manifest the siRNA effect. This piggy-back mechanism may be responsible for reported deep tissue siRNA effects using certain carriers.     

Smoke gas analysis by Fourier transform infrared spectroscopy – summary of the SAFIR project results

The determination of toxic components from fire gases is difficult because the environment is hot, reactions are often temperature dependent, and a lot of soot may be produced. Due to the different properties of the gas components, a different time‐consuming procedure for each species has traditionally been used. The use of FTIR (Fourier transform infrared) spectrometers as a continuous monitoring technique overcomes many of the problems in smoke gas analyses. FTIR offers an opportunity to set up a calibration and prediction method for each gas showing a characteristic spectral band in the infrared region of the spectrum. The objective of the SAFIR project was to further develop the FTIR gas analysis of smoke gases to be an applicable and reliable method for the determination of toxic components in combustion gases related to fire test conditions. The optimum probe design, filter parameters and the most suitable sampling lines in terms of flow rate, diameter, construction material and operating temperature have been specified. In the large scale, special concern was given to the probe design and the effects of the probe location as well as practical considerations of the sampling line length. Quantitative calibration and prediction methods have been constructed for different components present in smoke gases. Recommendations on how to deal with interferents, non‐linearities and outliers have been provided and a verification method for the spectrometer for unexpected variations and for the different models have been described. FTIR measurement procedures in different fire test scenarios have been studied using the recommendations of this project for measurement techniques and analysis and an interlaboratory trial of the FTIR technique in smoke gas analysis was carried out to define the repeatability and reproducibility of the method in connection with a small scale fire test method, the cone calorimeter. Copyright © 2000 John Wiley & Sons Ltd.     

Fluorescence characteristics of thermo-altered exinites (sporinites)

Fluorescence spectra of heat-treated exinites (sporinites) vary systematically with increasing temperatures of treatment. The wavelength of the fluorescence maximum, λ_max, shifts toward the longer wavelength as a result of thermal alteration. The maxima shift from 560 nm in untreated lignite to 640 nm in lignite after heating to 350 °C. Numerical values, such as fluorescence maximum, λ_max, and spectral quotient, $\text{Q}{}_{\mathrm{S}}\text{=}\text{I}{}_{650}{}_{\mathrm{I}}\text{500}$, can be used as rank parameters of coals. The technique can also be used as a tool to monitor thermal reactions of coals at relatively moderate temperatures. The method can distinguish certain resinites from exinites.     

Decontamination of petroleum sludges by hot water extraction

The process of hot water extraction of tar sand was modified and adapted for removal of heavy oil from bottom tank petroleum sludges, and was submitted to a laboratory feasibility study. This process can also be utilized to clean beach sands contaminated by accidental heavy oil spills. In the case of oil contaminated sands, a single stage extraction has yielded a 99% recovery of hydrocarbons and clean sands (containing less than 0.1% of hydrocarbons) which are thus safe to be returned to the environment. In the case of petroleum bottom tank sludge, it was necessary to proceed with a double stage extraction with the addition of a wetting agent. A dosage of a Na₂ Si O₃ aqueous solution of 1% by weight has proven efficient, allowing an 82% recovery of hydrocarbons, with only 0.5% of hydrocarbons content in the solid residues.